Small cationic antimicrobial peptides delocalizeperipheral membrane proteinsMichaela Wenzela, Alina Iulia Chiriacb, Andreas Ottoc, Dagmar Zweytickd, Caroline Maye, Catherine Schumacherf,Ronald Gustg, H. Bauke Albadah, Maya Penkovah, Ute Krämeri, Ralf Erdmannj, Nils Metzler-Nolteh, Suzana K. Strausk,Erhard Bremerl, Dörte Becherc, Heike Brötz-Oesterheltf, Hans-Georg Sahlb, and Julia Elisabeth Bandowa,1

aBiology of Microorganisms, hBioinorganic Chemistry, and iPlant Physiology, eImmune Proteomics, Medical Proteome Center, and jInstitute of PhysiologicalChemistry, Ruhr University Bochum, 44801 Bochum, Germany; bInstitute for Medical Microbiology, Immunology, and Parasitology, PharmaceuticalMicrobiology Section, University of Bonn, 53113 Bonn, Germany; cDepartment of Microbial Physiology and Molecular Biology, Ernst Moritz Arndt University,17489 Greifswald, Germany; dBiophysics Division, Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria; fInstitute for PharmaceuticalBiology and Biotechnology, Heinrich Heine University, 40225 Düsseldorf, Germany; gDepartment of Pharmaceutical Chemistry, University of Innsbruck, 6020Innsbruck, Austria; kDepartment of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1; and lDepartment of Biology, University ofMarburg, 35037 Marburg, Germany

Edited by Michael Zasloff, Georgetown University Medical Center, Washington, DC, and accepted by the Editorial Board February 27, 2014 (received forreview October 22, 2013)

Short antimicrobial peptides rich in arginine (R) and tryptophan(W) interact with membranes. To learn how this interaction leadsto bacterial death, we characterized the effects of the minimalpharmacophore RWRWRW-NH2. A ruthenium-substituted deriva-tive of this peptide localized to the membrane in vivo, and thepeptide also integrated readily into mixed phospholipid bilayersthat resemble Gram-positive membranes. Proteome and Westernblot analyses showed that integration of the peptide caused de-localization of peripheral membrane proteins essential for respi-ration and cell-wall biosynthesis, limiting cellular energy andundermining cell-wall integrity. This delocalization phenomenonalso was observed with the cyclic peptide gramicidin S, indicatingthe generality of the mechanism. Exogenous glutamate increasestolerance to the peptide, indicating that osmotic destabilizationalso contributes to antibacterial efficacy. Bacillus subtilis respondsto peptide stress by releasing osmoprotective amino acids, in partvia mechanosensitive channels. This response is triggered by mem-brane-targeting bacteriolytic peptides of different structural clas-ses as well as by hypoosmotic conditions.

mechanism of action | respiratory chain | hypoosmotic stress response |metallocenes

During the past two decades, bacterial antibiotic resistance hasgrown into a major threat to public health, restoring in-

fectious diseases to the list of leading causes of death worldwide(www.who.int/healthinfo/statistics/bodgbddeathdalyestimates.xls).As a reaction to this health crisis, several efforts currently areunderway to discover and develop new natural and synthetic an-tibiotic compounds. One promising antibiotic class is antimicrobialpeptides, which occur naturally as part of host defense systems inall domains of life (1–4). Antimicrobial peptides range in lengthfrom four to more than 100 amino acids and fall into a number ofdifferent structural classes, including α-helical amphiphiles, lipo-peptides, glycopeptides, lantibiotics, and short cationic peptides.The short cationic peptides offer a potent alternative to longer

natural antimicrobial peptides. The latter can be difficult toisolate and are complicated to synthesize chemically, but shortcationic peptides are generated readily by solid-phase peptidesynthesis and are easily accessible for chemical derivatization(5–8). They are characterized by positively charged and byhydrophobic amino acids (9, 10). Previous mechanistic studiesin vitro have examined the interactions of the peptides withmembranes or membrane extracts (11–15), but their effects onbacterial physiology have been largely underexplored.A more complete understanding of how these short cationic

antimicrobial peptides bring about bacterial cell death is neededto further their optimization and development for practicalapplications. To achieve this understanding, we studied the

synthetic hexapeptide RWRWRW-NH2 (referred to hereafter as“MP196”) (see SI Appendix, Fig. S1 for structure) as a modelrepresenting the minimal pharmacophore of positively chargedand hydrophobic amino acids (16, 17). It is effective against Gram-positive bacteria including methicillin-resistant Staphylococcusaureus strains, is moderately effective against Gram-negative bac-teria, has low toxicity against human cell lines, and displays lowhemolytic activity (18). MP196 therefore is a promising leadstructure for derivatization and already has yielded peptides withimproved activities and altered pharmacological properties (19).For example, in a recent systematic L-to-D exchange scan peptideswith significantly reduced hemolytic activity could be identified (20).Proteomic profiling of the Gram-positive bacterium Bacillus

subtilis exposed to MP196 provided a starting point for mechanistic

Significance

Multidrug-resistant bacteria present an acute problem tomedicine, generating interest in novel antimicrobial strategies.Antimicrobial peptides currently are being investigated, both asantibiotics and as immunomodulatory agents. Many antimicro-bial peptides interact with the bacterial membrane, a previouslyunderexplored antibiotic target. We present a system-based studyof the mode of action of small cationic peptides and the mecha-nisms that bacteria use to defend against them. We show thatpeptide integration into the membrane causes delocalizationof essential peripheral membrane proteins. This delocalizationimpacts on two cellular processes, namely respiration and cell-wall biosynthesis. We describe a bacterial survival strategy inwhich mechanosensitive channels in the bacterial membraneestablish osmoprotection against membrane-targeting bacte-riolytic peptides. Understanding the peptides’ mode of actionand bacterial survival strategies opens up new avenues fordevising peptide-based antibacterial strategies.

Author contributions: M.W., H.-G.S., and J.E.B. designed research; M.W., A.I.C., A.O., D.Z.,C.M., C.S., and R.G. performed research; H.B.A., M.P., U.K., N.M.-N., S.K.S., E.B., D.B., andH.B.-O. contributed new reagents/analytic tools; M.W., A.I.C., A.O., D.Z., C.M., C.S.,R.G., R.E., H.B.-O., and J.E.B. analyzed data; and M.W., H.B.-O., H.-G.S., and J.E.B. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. M.Z. is a guest editor invited by theEditorial Board.

Data deposition: The mass spectrometry proteomics data have been deposited to theProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via thePRIDE partner repository [Vizcaino JA, et al. (2013) The Proteomics Identifications (PRIDE)database and associated tools: Status in 2013. Nucleic Acids Res 41(D1):D1063–D1069].The dataset ID is PXD000181.1To whom correspondence should be addressed. E-mail: julia.bandow@rub.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319900111/-/DCSupplemental.

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studies. It identified two major areas for analysis: membrane andcell-wall integrity and energy metabolism. The proteomeanalysis prompted further investigation into the deregulation ofamino acid biosynthesis, which revealed that B. subtilis coun-teracts the attack on the cell envelope by triggering an osmo-protective release of glutamate.

ResultsInhibition of Macromolecule Biosynthesis Points to the Bacterial CellEnvelope as a Target of MP196. To explore the inhibition of mac-romolecule biosynthesis by MP196 in vivo, we studied incorporationof radioactive precursors. Cells were exposed to MP196 concen-trations that reduced growth rates by ∼50% unless noted otherwise(SI Appendix, Fig. S2). MP196 moderately inhibited incorporationof DNA, RNA, protein, and cell-wall precursors (Fig. 1A andSI Appendix, Fig. S3). To separate the primary event from sec-ondary effects, the activation of reporter genes was studied inB. subtilis (Fig. 1B). Each of the reporter strains had been shownpreviously to respond specifically to the inhibition of a particularcellular process using different antibiotics (21). MP196 did notactivate transcription from promoters of yorB, helD, or bmrC,which are indicative of DNA damage, RNA synthesis inhibition,and translation inhibition, respectively. The liaI promoter, whichis responsive to inhibition of steps in membrane-bound cell-wallbiosynthesis, was activated weakly, whereas ypuA, which is re-sponsive to both membrane-associated and membrane-independentcell-wall stress, was strongly activated.

The Proteomic Response of B. subtilis Provides Additional EvidenceThat MP196 Targets the Cell Envelope. The response of B. subtilis totreatment with MP196 was studied at the proteome level bycombining 2D gel-based and LC-MS–based approaches (SI Ap-pendix, Results of the Comparative Proteome Analysis and Tables

S1–S4). Proteins indicative of general cell-envelope stress, mem-brane stress, energy limitation, and cell-wall stress were stronglyup-regulated upon exposure to MP196 (Fig. 1C), indicating thatthe cell envelope is the primary target of MP196. From a libraryof proteome response profiles that includes the responses tomore than 50 antibiotic agents (22–26), several antibiotics wereidentified that share subsets of these marker proteins withMP196, all affecting the bacterial cell envelope (SI Appendix,Table S5). The strongest overlap in up-regulated proteins wasobserved for the membrane-targeting agents gramicidin S andvalinomycin. Gramicidin S, a member of the small cationic an-timicrobial peptide class, is structurally distinct from MP196: Itis a cyclic amphipathic peptide that does not contain the ar-ginine or tryptophan residues found in MP196. It disruptsmembrane function by integrating into the lipid bilayer (27).Valinomycin is an uncharged dodecadepsipeptide, which acts asa potassium-carrier ionophore. Both antibiotics were selected asclose comparators to study the antibiotic mechanism of MP196.We also chose vancomycin and nisin as comparator compounds.They both share marker proteins of cell-envelope stress withMP196. Vancomycin is a lipid II-binding cell-wall biosynthesisinhibitor, and nisin, in addition to inhibiting cell-wall bio-synthesis by binding lipid II, forms heteromultimeric membranepores with lipid II.

A Stereospecific Binding Site Is Not Required for MP196 TargetInteraction. Given the proteomics results, it seemed likely thatthe lipid bilayer is a direct target of the peptide. However, toexclude the possibility that MP196 binds to a protein target anddownstream effects elicit the observed proteomic response, weinvestigated the importance of peptide stereochemistry for an-tibacterial activity and MP196–target interaction using an all–D-amino acid peptide (D-MP196). D-MP196 was as active as MP196

Fig. 1. Narrowing down the target area. (A) [3H]-glucosamine incorporation by S. simulans upon treatment with MP196 or vancomycin. (B) Transcriptionactivation of selected B. subtilis promoters indicative of inhibition of specific cellular processes. Cells were incubated with increasing concentrations of MP196for predetermined times depending on the induction kinetics of the respective reporter strains. (C) 2D gel-based proteome analysis of MP196-treatedB. subtilis. Synthesis rates of cytosolic proteins of MP196-treated (false-colored in red) and untreated (false-colored in green) B. subtilis were compared basedon [35S]-methionine labeling. In the overlaid autoradiographs, down-regulated proteins appear green, up-regulated proteins appear red, and proteinsexpressed at equal rates appear yellow. Unidentified proteins are marked by circles. Blue labels indicate marker proteins for general cell-envelope stress,green labels identify cell-wall biosynthesis inhibition, and red labels are markers for membrane stress.

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against B. subtilis (18), and the two forms had highly similarproteome responses (SI Appendix, Fig. S4 A and B). Thus,stereochemistry is not crucial for target interaction, making itunlikely that MP196 binds to a specific site on a protein target.

MP196 Affects Cell Morphology. The impact of nonlytic concen-trations of MP196 and gramicidin S on the morphology ofB. subtilis cells was examined by transmission electron microscopy(TEM) (Fig. 2A). MP196-treated cells displayed characteristiccell-wall lesions. The cell wall at these sites is thinner, suggestinga loss of integrity. Gramicidin S-treated cells did not retain theirshape during sample preparation, culminating in the leakage ofcell contents and cell collapse. Thus, although both peptides in-duce similar proteome-response patterns, the structural impair-ment of the B. subtilis cell envelope is more severe after gramicidinS treatment than after MP196 exposure.

MP196 Accumulates in the Bacterial Cell Envelope. To study thesubcellular localization of MP196 in vivo, a ruthenocene-substituted derivative Rc-C(O)-WRWRW-NH2 (MP276) (see SIAppendix, Fig. S1 for structure) was synthesized for TEM andatomic absorption spectrometry studies. The ruthenocene sub-stitution did not affect antibacterial activity negatively (18), andruthenium alone is not toxic.Thin sections of untreated and MP276-treated B. subtilis were

prepared omitting the lead-staining step, which is used as con-trast agent in electron microscopy (Fig. 2B). Accordingly, in thepreparations of untreated cells the cell envelope is poorly con-trasted compared with the cytosol. However, in MP276-treatedcells the cell envelope is seen in high contrast, showing that thepeptide with the electron-dense ruthenium label accumulates inthe cell envelope.

Peptide Concentrations Are Highest in the Bacterial Membrane.Graphite furnace atomic absorption spectrometry provides ab-solute quantitation of the ruthenium-labeled peptide in subcellularfractions. Because ruthenium does not occur naturally in bacteria, itis ideally suited for peptide tracing in the cellular environment (28).The absolute ruthenium amounts were related to the volumes

of the cellular compartments to give compartment concentrations(SI Appendix, Table S6). Volumes were calculated based on high-resolution cryo-electron microscopy studies of the B. subtilismembrane and cell-wall structures (29, 30). The ruthenium con-centration in the membrane fraction exceeded concentrationsmeasured in the cytosolic and cell-wall fractions by factors of 15and 17, respectively. In agreement with peptide tracing by TEM,the principal localization of MP276 is the membrane. Duringsample preparation, cells were washed with EDTA several times.There was no significant elution of ruthenium (∼10−21 mol percell), suggesting strong binding of the peptide to the phospholipidbilayer. Lower but still significant amounts of peptide weredetected in the cell wall, which can function as a barrier thatdetains antibiotics (31). Concentrations of ruthenium above back-ground levels also were detected in the cytosolic fraction, sug-gesting that the peptide has some ability to cross the lipid bilayer.

MP196 Integrates into Phospholipid Bilayers That Mimic Gram-Positive Membranes but Not Erythrocyte Membranes. Interactionof MP196 with the membrane was investigated by differentialscanning calorimetry (DSC) using model membranes consistingof the two most abundant phospholipids in the B. subtilismembrane (32), namely 1,2-dipalmitoyl-sn-glycero-3-phospha-tidylglycerol (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phos-phatidylethanolamine (DPPE), in an 88:12 ratio (Fig. 3A). DSCmonitors the thermotropic-phase behavior of the lipid bilayer.Changes in pattern are indicative of perturbation of the mem-brane bilayer and packaging of the phospholipids’ fatty acylchains and, therefore, of peptide integration. Deconvolution of

the thermograms indicates that MP196 integrates into the phos-pholipid bilayer, generating “peptide-affected domains” (14).These are characterized by a broadening of the existing transitionand the appearance of a small transition at lower temperatures,reflecting rearrangement of the lipid mixture, and, consequently,alterations in packaging of the lipids at the emerging borderlines.When bilayers composed of a single type of phospholipid were

used, the influence of MP196 was far less pronounced. Abroadening of the shoulder at lower temperatures indicated theformation of some peptide-affected domains on DPPG bilayers,

Fig. 2. Peptide effects on cells and subcellular localization. (A) TEM imagesof peptide-treated B. subtilis. Cells were fixed with 2% uranyl acetate, andultrathin sections were stained with 0.2% lead citrate in 0.1 M NaOH. (B) Invivo peptide tracing using the ruthenocene-substituted MP196 derivativeMP276. B. subtilis cells were fixed with 2% uranyl acetate. Sections remainedunstained to avoid interference of lead with the ruthenium signal. Ruthe-nium in the cytosol, membrane, and cell wall was quantified by atomic ab-sorption spectroscopy.

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but no effect was seen using pure DPPE (SI Appendix, Fig. S5) orpure 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)(Fig. 3B), which typically are used to model erythrocyte mem-branes (33). That MP196 affected DPPG but not DPPE impliesa preference for negatively charged over neutral lipids and couldexplain the preference for bacterial over erythrocyte membranes.This supposition is consistent with the known low hemolytic ac-tivity of MP196 (18). A preference for negatively charged phos-pholipids has been shown previously for other very small RW-rich peptides, Combi-1 (Ac-RRWWRF-NH2) and LfcinB4–9(RRWQWR-NH2), using the same method (reviewed in ref. 13).

Peptide Integration Does Not Lead to Pore Formation. We usedfluorescence staining to test whether in vivo integration of theMP196 peptide results in pore formation in B. subtilis. The green-fluorescent dye SYTO 9 crosses intact membranes, whereas thered-fluorescent propidium iodide enters bacterial cells throughlesions in the membrane (Fig. 3C). Pore-forming nisin allowedboth dyes to enter B. subtilis cells, resulting in orange cells. MP196treatment, as well as treatment with non–pore-forming gramicidinS and valinomycin, resulted in green-fluorescent cells—like theuntreated control—suggesting that MP196 does not act by form-ing large pores. The same conclusions were drawn from measuringpeptide-induced potassium leakage from Bacillus megateriumcells into choline buffer (SI Appendix, Fig. S6A). In contrast tothe positive control, nisin, MP196 released little potassium, evenat concentrations equivalent to 50 times the minimal inhibitoryconcentration (MIC).

MP196 Does Not Disturb Metal Cation Homeostasis. The B. subtilisproteomic responses to MP196 and to the potassium ionophorevalinomycin were markedly similar. To investigate whetherMP196 integration into the membrane disturbs metal cationhomeostasis, total cellular ion concentrations were determinedafter peptide treatment in chemically defined culture broth (SIAppendix, Fig. S6B). In agreement with the observed lack ofpotassium efflux in choline buffer, no significant decrease in anymetal ion was observed after MP196 treatment. In contrast,gramicidin S treatment caused a significant decrease in potas-sium, magnesium, and manganese levels. Valinomycin-treatedcells selectively accumulated potassium (an eightfold increase),probably because of overcompensation. These results fit wellwith the observation that, despite provoking a strong proteomeresponse, valinomycin does not inhibit B. subtilis growth (24).

MP196 Integration Causes Membrane Depolarization. Many mem-brane-targeting antibiotics, including gramicidin S and valinomy-cin, depolarize the bacterial membrane (27, 34). Depolarizationwas investigated using a B. subtilis strain carrying a GFP fusion tothe cell division-regulating protein MinD. Normally localized atthe cell poles and in the cell-division plane, MinD delocalizesupon depolarization, resulting in a spotty pattern of GFP-MinDdistribution (35). In contrast to the normal MinD pattern in theuntreated control cells, MinD delocalization was seen in MP196and gramicidin S-treated cells (Fig. 4A). Depolarization uponMP196 treatment was confirmed in B. megaterium with the volt-age-sensitive fluorescent probe DiSC35 (SI Appendix, Fig. S6C).

ATP Levels Drop in MP196-Treated Cells. Sudden depolarizationshould result in energy limitation, and energy limitation alsowould be consistent with the proteome response. IntracellularATP levels of B. subtilis, determined using a luciferase assay,showed a 60% reduction in cellular ATP content in MP196-treated cells and a 70% reduction in gramicidin S-treated cells(Fig. 4B), whereas valinomycin and nisin caused 30% drops.Thus, MP196 has a considerable impact on energy metabolism.No significant ion efflux was observed under the test conditions,indicating that depolarization and energy limitation are not causedsolely by ion transfer.

MP196 Inhibits the Respiratory Chain at the Cytochrome c Level.Because of the lack of pore formation and ion transfer, whichare typical causes of a breakdown of energy metabolism, we testedwhether components of the respiratory chain are inhibited byMP196. Inhibition of ATP synthase would limit ATP directly.Proton-pumping activity was monitored in Micrococcus flavusinverted vesicles using the pH-sensitive probe acridine orange(SI Appendix, Fig. S6D). The ATP synthase inhibitor lacto-ferrin (36) served as a positive control. No inhibition of ATPsynthase activity was observed at MP196 concentrations cor-responding to those used in the proteome analysis.Inhibition of the respiratory chain upstream of ATP synthase

would contribute to the breakdown of the membrane potential,resulting in the loss of ATP synthesis. Using M. flavus invertedvesicles, inhibition of the respiratory chain was monitored withiodonitrotetrazolium chloride, a reduction-sensitive dye (Fig.4C). Rotenone and antimycin A, specific inhibitors of complexesI and III of the respiratory chain, respectively, were used ascomparator compounds. Rotenone reduced electron transportactivity by 20%, and antimycin A did so by 50%. MP196 dis-played similar inhibition efficiency, reducing electron transportby 30%. MP196 inhibition was additive with either rotenone orantimycin A, suggesting that MP196 inhibits a different compo-nent of the respiratory chain.Cytochrome c, which is located on the outer membrane leaflet,

transfers electrons from complex III to complex IV. Its locali-zation after MP196 treatment was investigated by Western blot

Fig. 3. Interaction with the membrane. (A) DSC thermograms of lipidbilayers consisting of 88:12 DPPG/DPPE incubated with MP196. Changes inthermotropic-phase behavior caused by perturbation of fatty acyl packingindicate peptide integration. (B) DSC thermograms of lipid bilayers con-sisting of DPPC. (C ) Overlaid fluorescence microscopy images of Bac-Light-stained B. subtilis treated with valinomycin, nisin, gramicidin S, andMP196. A red-fluorescing dye selectively stains cells with large membraneholes; a green-fluorescing dye stains all cells independently of membraneintegrity. Cells with intact membranes appear green, and cells with perfo-rated membranes appear orange.

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analysis of membrane fractions of antibiotic-treated B. subtilis cells(Fig. 4D). The amount of cytochrome c in the membrane wasreduced drastically after 5 min of treatment with MP196, in-dicating dissociation of the protein from the extracellular mem-brane surface. This delocalization is independent of the membranepotential breaking down, because cytochrome c was detected inthe membrane fraction of the untreated control cells, which weredisrupted by ultrasonication. Rather, delocalization requires in-teraction of the peptide with the membrane. A similar effect wasobserved using gramicidin S, which previously was shown to inhibitcomponents of the respiratory chain (37). Detachment of cyto-chrome c from the outer membrane leaflet explains the inhibitionof the respiratory chain, breakdown of the membrane potential,and subsequent energy limitation (Fig. 4E) (38).

MP196 Undermines Cell-Wall Integrity by Delocalizing AnotherEssential Peripheral Membrane Protein. Following MP196 expo-sure, cell-wall biosynthesis is slightly inhibited (as revealed byprecursor incorporation), severe cell-wall lesions form (as revealedby TEM), and there is a significant overlap of marker proteinswith compounds that target membrane-bound steps of cell-wallbiosynthesis in proteomic studies. When incorporation of cell-wallmaterial is inhibited at a step in membrane-bound biosynthesis,B. subtilis cells undergo a characteristic change in shape afteracetic acid/methanol fixation, namely extrusion of the cell mem-brane through holes in the cell wall (4, 26). Untreated cells andcells treated with valinomycin did not display membrane extru-sions (Fig. 5A); nisin and MP196 did induce membrane extrusions,

as did gramicidin S (although gramicidin S is not known to in-terfere directly with cell-wall biosynthesis).A stereospecific target interaction was excluded by experi-

ments with the D-MP196 variant. Therefore we investigatedwhether inhibition of a membrane-bound step of cell-wall bio-synthesis could be attributed to the delocalization of a peripheralmembrane protein, similar to the inhibition of the respiratorychain by cytochrome c delocalization. In B. subtilis, the onlyperipheral membrane protein in the pathway is MurG, the en-zyme that converts lipid I to lipid II by attaching GlcNAc to thebactoprenol carrier-conjugated UDP-N-acetylmuramic acid(UDP-MurNAc) pentapeptide molecule. When the localizationof MurG was investigated by Western blot analysis (Fig. 5B),both MP196 and gramicidin S treatment caused almost completeloss of MurG from the membrane fraction of treated B. subtiliswithin 5 min. The removal of MurG from the cytosolic mem-brane surface, in combination with energy limitation (see above),may explain the reduced glucosamine incorporation, loss of cell-wall integrity, and induction of a cell-wall–specific stress response.Inhibition of earlier cytosolic steps of cell-wall biosynthesis, in-hibition of membrane-bound steps by lipid II binding, and directinhibition of MurG activity were excluded experimentally (SIAppendix, Results of Cell Wall Biosynthesis Inhibition Assaysand Fig. S7).

MP196 Integration Triggers MurG Release from Isolated B. subtilisMembranes. Western analysis of disrupted cell membranes revealedthat both cytochrome c and MurG bind the membrane independent

Fig. 4. Effects on membrane potential and respiration. (A) Fluorescence microscopy images (Upper Row) and corresponding bright-field images (Lower Row)show GFP-MinD localization in untreated B. subtilis cells and cells incubated with gramicidin S or MP196. GFP-MinD delocalization indicates membranedepolarization. (B) ATP levels of B. subtilis cells treated with valinomycin, nisin, gramicidin S, or MP196. In a luciferase assay, ATP concentrations were de-termined under conditions resembling those of the proteome experiment. (C) Activity of the respiratory chain of inverted M. flavus vesicles treated withrotenone, antimycin A, or MP196. Electron transport efficiency was monitored by the reduction of iodonitrotetrazolium chloride causing a decrease inabsorbance at 485 nm. (D) Western analysis of cytochrome c in membrane fractions of B. subtilis after a 5-min treatment with gramicidin S and MP196. ThePonceau S-stained blot is displayed as loading control. (E) Overview of the bacterial respiratory chain [according to Vonck and Schäfer (38)]. Proton move-ments are indicated by green arrows; electron movements are indicated by orange arrows.

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of the membrane potential. To test if MP196 integration is sufficientfor the release of MurG from the membrane, the membrane frac-tion of disrupted B. subtilis was collected and incubated with thepeptide in vitro (Fig. 5C). Western blot analysis showed that MurGwas released efficiently from the MP196-treated membrane fractionbut remained attached to the untreated membrane, demonstratingthat MP196 is sufficient for MurG detachment from the membrane.

B. subtilis Counteracts MP196 Action by Osmotic Stabilization. Pro-teome analysis revealed up-regulation of proteins involved inamino acid anabolism, especially of glutamate and aspartate,within 65 min of MP196 treatment (SI Appendix, Table S3). Toinvestigate the influence of MP196 on the amino acid pool, in-tracellular and extracellular free amino acid levels were mea-sured by HPLC after 15 min of antibiotic treatment. MP196-treated cells accumulated free arginine and valine intracellularly(Fig. 6A and SI Appendix, Fig. S8A), consistent with the up-regulation of proteins involved in valine biosynthesis (SI Ap-pendix, Table S3). Intracellular glutamine/glutamate, asparagine/aspartate (the assay used here does not distinguish betweenglutamate and glutamine or between aspartate and asparagine),lysine, and proline levels were substantially reduced, with con-comitant dramatic increases in extracellular glutamine/glutamateand asparagine/aspartate and moderate increases in extracel-lular arginine, lysine, and proline (Fig. 6B and SI Appendix,Fig. S8B). The absolute amounts of glutamine/glutamate re-leased exceeded by far the sum of intracellular and extracellularlevels of untreated B. subtilis cultures, indicating dedicated bio-synthesis and export (SI Appendix, Fig. S8C).Supplementing defined medium with exogenous glutamate

increased the MIC of MP196 against B. subtilis by up to eightfold(Fig. 6C), establishing that glutamate has a protective effectagainst MP196-induced stress. Similar results were obtained bysupplementation with exogenous sodium chloride (Fig. 6D) andpotassium chloride (Fig. 6E), suggesting that the protection againstMP196 is not specific for glutamate but is caused by osmotic effects.Glutamate release also was observed in response to osmotic

downshift and treatment with bacteriolytic peptides that target themembrane: the cyclic and cationic gramicidin S, the α-helical cat-ionic aurein 2.2, the cationic lantibiotic nisin, or the unchargedamphiphilic gramicidin A. It was not observed after osmotic upshiftor treatment with valinomycin or vancomycin, peptides that areeither not bacteriolytic or do not target the membrane (Fig. 6F).Mechanosensitive channels appear to contribute to glutamaterelease after MP196 treatment. A B. subtilis mutant that lacksthe four known MscL and MscS-type mechanosensitive channels(39) accumulated high levels of glutamate intracellularly (Fig.6G) and showed heightened sensitivity to MP196 (Fig. 6 C–E).However, these channels are not the only route for glutamaterelease, because extracellular glutamate levels increased whenthe quadruple msc mutant was treated with MP196 (Fig. 6B).

DiscussionThe cationic hexapeptide MP196, which is the minimal phar-macophore of RW-rich peptides, was used here to study themechanism of action of short cationic antimicrobial peptidesand subsequent bacterial adaptation strategies. MP196 acts onthe bacterial cytoplasmic membrane (SI Appendix, Fig. S9 A, I),where essential physiological processes, including respiration (SIAppendix, Fig. S9 A, II), cell-wall biosynthesis (SI Appendix, Fig.S9 A, III), and membrane transport SI Appendix, Fig. S9 A, IV),take place. At the molecular level (SI Appendix, Fig. S9 B, I),MP196 integrates into the lipid bilayer readily and disturbsmembrane architecture, as revealed by the perturbation of fattyacyl packing in the DSC analysis. This membrane integration likelyis promoted by the interaction of the cationic arginine residueswith negatively charged phospholipid head group and is facilitatedby lipophilic tryptophan residues. Such a mechanism of membraneintegration is typical of amphipathic peptides (13, 40–42), whichaccumulate in the membrane near the interface with the cytosol,making contact with lipid head groups and fatty acyl chains.RW-rich peptides have been shown to differ in their ability to

permeabilize membranes. Although the tridecapeptides tritrpti-cin and indolicidin permeabilize lipid bilayers (43), hexapeptidessuch as Combi-1 and Combi-2 showed very poor permeabiliza-tion (44). It is important to note that even peptides with verysmall structural differences can have distinct mechanisms ofinteracting with membranes, resulting in different permeabiliza-tion capabilities and modes of action, as shown recently for verysmall cyclic RW-rich peptides (45). Thus, the observationsnoted in this study may not necessarily apply to all antimicro-bial peptides.For MP196 we found no evidence for membrane permeabiliza-

tion or pore formation in model membranes or in vivo. No ionrelease occurs in chemically defined bacterial culture medium. Ithas been suggested that integration of cationic amphiphatic pep-tides leads to local lipid reorganization and the formation of“peptide-induced lipid pores” that allow metal ions to cross themembrane (42); however, the lack of ion release in chemically de-fined medium suggests that ion efflux is not a major effect of MP196under the growth conditions used in the proteome experiment.The integration of MP196 into the membrane affects multiple

cellular processes (SI Appendix, Fig. S9 B, II–IV). Like the RW-rich small cationic antimicrobial peptide Bac8c (46) and thesmall cyclic cationic peptide gramicidin S (37) that does notcontain arginine or tryptophan residues, MP196 has a profoundimpact on the bacterial respiratory chain (SI Appendix, Fig. S9 B,II). Our data suggest that the inhibition of the respiratory chainby small cationic antimicrobial peptides is caused by the de-tachment of cytochrome c from the membrane, resulting indisruption of the electron transfer chain and subsequent break-down of the proton gradient. Inhibition of the respiratory chainthen affects ATP synthesis, causing energy limitation thatimpacts all other macromolecule biosynthesis pathways.

Fig. 5. Effects on the cell wall. (A) Light microscopy images showing cell-wallintegrity of B. subtilis after treatment with MP196 and nisin. Acetic acid-methanol fixation visualizes cell-wall damage by extrusions of the membranethrough holes in the cell wall. (B) Western blot detection of the cell-wallbiosynthesis protein MurG in membrane fractions isolated from B. subtiliscells that were treated with peptide for 5 min. (C) Western blot detection ofMurG in B. subtilismembrane fractions that first were isolated and then wereincubated with peptide for 5 min.

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The impact of MP196 on cell-wall biosynthesis is illustrated bya reduction in precursor incorporation, promoter activation ofcell-wall stress-responsive genes, up-regulation of proteins in-dicative of cell-wall biosynthesis stress, and loss of cell-wall in-tegrity. Similar observations have been reported for gramicidin S(26) and Bac8c (46). In addition to an indirect effect on cell-wallbiosynthesis caused by energy limitation, we found that smallcationic peptides affect cell-wall biosynthesis by delocalizing thelipid II biosynthesis protein MurG from the intracellular surfaceof the membrane. As with cytochrome c, protein delocalization isindependent of the membrane potential. Rather, it seems to bea consequence of the alterations in membrane architecturecaused by peptide integration, which were observed by DSC.Delocalization of MurG could explain the effects on cell-wallbiosynthesis and integrity exerted by MP196. This enzyme re-cently was shown to be the direct target of the anti-staphylo-coccal small molecule inhibitor murgocil (47).We suggest that delocalization of peripheral membrane pro-

teins is a general mechanism extending to membrane-targetingpeptides of other structural classes such as the lipopeptidedaptomycin. Daptomycin has been shown to alter membranearchitecture and to delocalize the cell-division protein DivIVC,which binds the membrane in a curvature-dependent fashion(48–50). Although delocalization of MurG by daptomycin hasnot been described thus far, it would explain the induction of thecell-wall stress response (51).A further consequence of MP196 treatment is the de-

localization of the cell-division regulating protein, MinD. MinDpreviously was shown to delocalize upon membrane depolarization(35). MinD and MurG attach to the intracellular surface of themembrane through electrostatic interactions using an amphipathic

helix motif (35, 52), whereas cytochrome c variants in B. subtilisattach to the outer leaflet of the membrane by a lipid anchoror transmembrane helices (53, 54). Thus, MP196 causes de-localization of proteins on both sides of the membrane, whichattach to the membrane by different mechanisms. It is possiblethat, in addition to the proteins studied here, other membrane-associated proteins are delocalized as a consequence of MP196-mediated membrane injury.Lacking evidence of a stereospecific binding site for MP196, no

specific receptor–ligand interaction is apparent from our studies.In fact, our data suggest that the molecular target of MP196 is thelipid bilayer itself, and no other specific target than the membraneis identified. However, the main functional impact of MP196 is onproteins located in the membrane. Therefore MP196 interferessimultaneously with a broad range of cellular processes, compli-cating the development of bacterial resistance. DSC experimentsshowed that the interaction of MP196 with the membrane criti-cally depends on lipid composition, thereby offering the oppor-tunity to design peptides selective for Gram-positive and Gram-negative organisms over mammalian cells.The proteome response and amino acid analysis provide evi-

dence that B. subtilis counteracts peptide-mediated membranestress by inducing three known defense strategies: adjustment ofmembrane lipid composition (55–57), stabilization of the mem-brane (58, 59), and restriction of access to the membrane by theenhancement of wall teichoic acid D-alanylation (SI Appendix,Fig. S9 C, I–III; see SI Appendix for details) (31, 60–62). Here wedescribe another survival strategy, one that entails the release ofselected amino acids from the cells (SI Appendix, Fig. S9 C, IV).Large quantities of glutamine/glutamate, plus significant amountsof asparagine/aspartate, arginine, proline, and lysine, were released

Fig. 6. Amino acid composition. (A) HPLC analysis of the intracellular amino acid composition of B. subtilis treated with MP196. (B) Extracellular amino acidcomposition of the same cultures. Only the amino acids whose concentrations changed significantly after peptide treatment are displayed here (see SIAppendix, Fig. S8 for full amino acid profiles). Amino acids are written in a one-letter code in the order of elution time from the column. Glutamate andglutamine as well as aspartate and asparagine are not distinguishable by this method and appear as one peak each. Tryptophan was not quantified here. (C–E) MICs of MP196 against B. subtilis 168, a B. subtilis strain lacking all known mechanosensitive channels (SMB80), and its parent strain (JH642) in definedmedium supplemented with increasing glutamate (C), NaCl (D), or KCl (E) concentrations. MICs were determined independently twice. (F) Intra- and ex-tracellular glutamate concentrations in B. subtilis cells under different antibiotic and osmotic stress conditions determined by amino acid analysis. (G) Intra-and extracellular glutamate concentrations in a B. subtilis JH642 and SMB80.

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into the medium, and biosynthesis of these amino acids wasreflected in the proteome response.The release of amino acids is mediated in part by mechano-

sensitive channels, which measure membrane tension, and theirtransient opening is triggered by turgor-induced pressure on thelipid bilayer (63). In plants, the activation of host defense bybacterial antimicrobial peptides has been attributed to mecha-noreceptors directly sensing peptide-induced alterations in mem-brane architecture (64–66).Glutamate is an osmoprotectant that accumulates intracellularly

in some bacilli. In B. subtilis it is the most abundant free amino acid,with a pool size of about 100 mM (67).Under hyperosmotic stress the intracellular glutamate pool

increases moderately (up to about 170 mM) (67). Under theseconditions the outflow of water is prevented by increasing pro-line levels (depending on the severity of the environmentallyimposed osmotic stress) up to pool sizes of 500 mM in severelystressed cells (68–70). Upon osmotic downshock the opposite isthe case: Escherichia coli releases potassium glutamate into themedium using mechanosensitive channels (71). In Corynebacteriumglutamicum glutamate excretion through mechanosensitive channelsis triggered in response to penicillin treatment (72). We show herethat B. subtilis also releases glutamate in response to hypoosmoticstress conditions. Glutamate release is triggered further in responseto treatment with membrane-targeting bacteriolytic peptides be-longing to different structural classes (cyclodecapeptide gramicidinS, α-helical amphiphile aurein 2.2, lantibiotic nisin, and unchargedamphiphilic gramicidin A) (Fig. 6F). These results suggest thatosmoprotective glutamate release is a general reaction to mem-brane-targeting bacteriolytic peptides. The addition of exogenousglutamate to the growth medium protected against MP196 via os-motic stabilization (Fig. 6 C–E). Similar observations were made forsublancin 168, a glycopeptide with an unknown mechanism of ac-tion produced by B. subtilis 168. High NaCl concentrations loweredthe susceptibility of sublancin-sensitive strains (73). High salt con-centrations also negatively affected the susceptibility of S. aureusstrains to the human antimicrobial peptides LL-37 (α-helical) andHNP-1 (β-sheet defensin) (74) as well as to the bactericidal activityof the small cationic peptide thrombin-induced platelet microbicidalprotein tPMP (75).This report provides a detailed physiological study of the in

vivo mechanism of action of a short RW-rich antimicrobial pep-tide, which we chose as a model to study the action of shortcationic antimicrobial peptides. It illustrates how MP196 inte-grates into the membrane, leading to delocalization of peripheralmembrane proteins. This delocalization includes the detachmentof the essential proteins cytochrome c and MurG from the cy-toplasmic membrane, impacting energy metabolism and cell-wallbiosynthesis. We propose that substantial energy limitation andloss of cell-wall integrity resulting from the delocalization ofessential peripheral membrane proteins are the major factorsthat contribute to MP196-mediated bacterial cell death. Theseperspectives on the action of small cationic antimicrobial pep-tides, involving lipids and proteins, are complementary to thelipid-focused pore formation and carpet mechanism models.Based on the data presented here, we suggest that MP196 actionfollows the interfacial activity model described by Wimleyand Hristova (11), whereby, at a reduction in growth rate of50%, phospholipid perturbation predominates over membranedisruption.We also describe a bacterial defense strategy against bacteri-

olytic membrane-targeting peptides that relies on lowering theconsiderable osmotic potential of the B. subtilis cell, and hencethe magnitude of turgor (67), through the release of selectedamino acids into the medium.

Materials and MethodsExperimental details and citations for all methods are provided in SI Appendix.

Radioactive Precursor Incorporation. The influence of MP196 on the majormacromolecular synthesis routes was studied by incorporation of radioac-tively labeled precursor molecules by Staphylococcus simulans as describedby Schneider et al. (76).

Reporter Gene Activation. Damage to the main cellular macromolecules wasmonitored by promoter activation of selected marker genes fused to thefirefly luciferase reporter gene in the genetic background of B. subtilis IS34.Serial twofold dilutions of each peptide (0.031–64 μg/mL) were inoculatedwith bacterial cell suspensions and incubated at 37 °C for a time perioddepending on the induction kinetics of the reporter strain. Then 2 mM lu-ciferin was added, and flash luminescence was measured.

Proteomics. Treatment of B. subtilis 168, metabolic labeling of newly syn-thesized proteins with [35S]-L-methionine, and subsequent protein separa-tion by 2D PAGE were performed as described previously (25). For gel-freeproteome analysis of membrane proteins, B. subtilis 168 was grown aero-bically at 37 °C in Belitzky minimal medium (BMM) supplemented with14N-ammonium sulfate and 14N-L-tryptophan. Cells were stressed at an OD500 of0.4 with 22.5 μg/mL MP196 for 65 min or were left untreated as control.Untreated cultures grown on 15N-ammonium sulfate and 15N-L-tryptophanwere used for relative quantification. Mixing of cell extracts before subcellularfractionation steps for relative quantification was carried out according toOtto et al. (77). The enriched membrane protein fraction was preparedaccording to the workflow published by Eymann et al. (78), omitting then-dodecyl-β-D-maltoside treatment step. The preparation of the integralmembrane peptides was carried out as described by Wolff et al. (79). Samplepreparation, mass spectrometric measurement, and subsequent data analysiswere carried out as described by Otto et al. (77). The mass spectrometry pro-teomics data have been deposited with the ProteomeXchange Consortium(http://proteomecentral.proteomexchange.org) via the PRIDE partnerrepository (80) with the dataset identifier PXD000181.

TEM. Cells were grown in BMM to an OD500 of 0.35. The main culture thenwas subdivided into 50-mL aliquots, and subcultures were treated with therespective antibiotics for 15 min or were left untreated as control. Cells wereharvested by centrifugation and washed twice in 100 mM Tris/1 mM EDTA,pH 7.5, and subsequently were washed once in the same buffer withoutEDTA. Bacterial samples for TEM were prepared according to Santhana Rajet al. (81) with some modifications (see SI Appendix, Experimental Details).To study effects on the cell envelope, sections were stained with 0.2% leadcitrate in 0.1 M NaOH for 3 s. Samples were examined at 23,000–230,000×magnification with a Philips EM410 transmission electron microscopeequipped with a Gatan digital camera system at an accelerating voltageof 80 kV.

For peptide tracing, cell cultures were treated with 1 μg/mL of the ruth-enocene-substituted MP196 derivative MP276 (SI Appendix, Fig. S3D) for15 min before sample preparation for TEM. The lead-staining step was omitted.

Graphite Furnace Atomic Absorption Spectrometry. Cytosolic, membrane, andcell-wall fractions of untreated and MP276-treated B. subtiliswere prepared.To this end, cells were harvested and washed five times with 100 mM Tris/1mM EDTA before ultrasonication, centrifugation, and ultracentrifugation.Ruthenium contents were quantified using a Vario 6 graphite furnace atomicabsorption spectrometer (Analytik Jena) as described previously (82–84).

Differential Scanning Calorimetry.Membrane integration was investigated byDSC-based determination of peptide-induced changes in the thermotropic-phase behavior of liposomes consisting of pure DPPG, pure DPPE, pure DPPC,and a mixture of 88% DPPG and 12% DPPE, respectively.

Permeability Assays. In vivo pore formation in B. subtilis 168 was monitoredusing the Live/Dead BacLight bacterial viability kit (Invitrogen) following themanufacturer’s instructions. Potassium release from B. megaterium wasperformed as described previously (26).

Global Ion Analysis. Ionomics experiments were performedwith B. subtilis 168in minimal medium under the same conditions as the proteomics experi-ments. Ion concentrations were determined by inductively coupled plasmaatomic emission spectroscopy.

Membrane Potential Measurements. GFP-MinD localization assays were per-formed with B. subtilis 1981 GFP-MinD (35) as described previously (26).

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Determination of membrane potential changes by use of DiSC35 was per-formed as described by Andrés and Fierro (36) in B. megaterium.

ATP Assay. ATP concentrations were determined from B. subtilis cytosolicextracts using the Perkin-Elmer ATPlite 1 step assay according to themanufacturer’s instructions.

ATPase and Respiratory Chain Assays. M. flavus inverted vesicles forH+-ATPase and respiratory chain activity measurements were preparedaccording to Burstein et al. (85). Proton pumping into M. flavus invertedvesicles by H+-ATPase was measured in a microtiter plate assay based onthe pH-sensitive probe acridine orange as described by Palmgren et al.(86). Antibiotic influence on the bacterial electron transport chain wasmonitored by reduction of iodonitrotetrazolium chloride using M. flavusinverted vesicles as described by Smith and McFeters (87). Cytochromec localization was determined by Western blot detection of cytochromec on the same blots used for MurG detection.

Cell-Wall Integrity and Inhibition of Biosynthesis. Sample preparation andbright field microscopic inspection of B. subtilis 168 was performed as de-scribed previously (26).

To detect MurG in membrane fractions of B. subtilis 168, cells were grownin BMM until early logarithmic growth phase, treated with antibiotics for 5min, and harvested by centrifugation. Membrane fractions were separatedby differential centrifugation, subjected to SDS PAGE, and blotted onto anitrocellulose membrane. MurG was detected with an E. coli MurG-specificantibody produced in rabbit and a secondary goat anti-rabbit IgG-HRPconjugate (BioRad).

Inhibition of in vitro lipid II synthesis was performed using M. flavus DSM1790 membrane preparations as described by Schneider et al. (76). For invitro lipid II binding, peptides were incubated with 2 nmol lipid II in 1:1molar ratios. Reaction mixtures were incubated at 30 °C for 30 min and

applied onto TLC plates (TLC Silica Gel 60 F254; Merck). Chromatography wasperformed in chloroform-methanol-water-ammonia (88:48:10:1) and stainedwith phosphomolybdic acid stain at 140 °C. Accumulation of UPP-MurNacpentapeptide was analyzed by HPLC as described by Schmitt et al. (88).

Amino Acid Analysis. Amino acid analysis of B. subtilis 168 (trpC2), B. subtilisJH642 (trpC2 pheA1), and SMB80 (trpC2 pheA1 mscL yhdY ykuT yfkC) (41)cellular extracts and culture supernatants was performed by HPLC using theAcquity HPLC and AccQ•Tag Ultra system (Waters GmbH) according to themanufacturer’s instructions. Before HPLC, proteins were removed byacetone precipitation.

MIC values against B. subtilis strains were determined with 5 × 105 cells/mLin BMM supplemented with increasing concentrations of glutamate, sodiumchloride, or potassium chloride. The lowest concentration inhibiting visiblegrowth was taken as the MIC.

ACKNOWLEDGMENTS. We thank Michaele Josten, Petra Düchting, MonikaBürger and Beate Menzel, and Stephanie Tautges, Kathrin Barlog, and JaleStoutjesdijk for excellent technical assistance; Christoph H. R. Senges for helpin preparing B. subtilis samples; Sina Langklotz and Dirk Albrecht for assis-tance with mass spectrometry; Helmut Meyer for providing amino acid anal-ysis technology; Klaus Funke and Ellen Kloosterboer for providing thecytochrome c antibody; Tanneke de Blaauwen and Franz Naberhaus for pro-viding the MurG antibody; and AiCuris, GmbH for providing the B. subtilisreporter strains. This manuscript was written in part at the 2013 ScientificWriting workshop of the Ruhr University Bochum (RUB) Research School; wethank Lars Leichert and Richard Gallagher for critically reading the manuscriptand for many helpful suggestions. This work was supported by a grant fromthe German federal state of North Rhine-Westphalia and the EuropeanUnion (European Regional Development Fund “Investing in your future”) (toN.M.-N., H.B.-O., H.-G.S., and J.E.B.) and by the RUB Research DepartmentInterfacial Systems Chemistry (N.M.-N. and J.E.B.). C.M. was supported by theProtein Unit for Research in Europe, a project of North Rhine-Westphalia.

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